Adeno-associated virus vector for boosting immunogenicity of freshly isolated tumor cells

ABSTRACT

The present invention provides an AAV vector having a foreign DNA coding for a protein that boosts immunogenicity of cells. The invention also provides a vaccine containing such a vector and the use of both.

This application is a continuation of U.S. application Ser. No.09/142,443, filed May 5, 1999 now U.S. Pat. No. 6,171,597; which was theNational Stage of International Application No. PCT/DE97/00445, filedMar. 6, 1997; which claims the benefit of a German Application No. 19608 751.1, filed Mar. 6, 1996.

FIELD OF THE INVENTION

The present invention relates to an adeno-associated virus vector suitedto increase the immunogenicity of cells, a vaccine containing such avector and the use of both.

BACKGROUND OF THE INVENTION

It is known that in about 0.5% of cancer patients, e.g., those sufferingfrom malignant melanomas, the tumor reverses completely. In many cancerpatients, a control of the tumor also takes place, so that it remains ina stable condition over years. This may be because the immune systeminfluences the course of the cancer.

Many attempts have been made to activate the immune system, and todetect and eliminate tumor cells. However, these attempts have not yetyielded satisfactory results.

It is an object of the present invention to provide a product by whichthe immune system can be stimulated with respect to tumor cells.

Adeno-associated viruses (AAVs) are single-stranded DNA virusesbelonging to the Parvovirus family. AAVs require helper viruses,particularly adenoviruses or herpesviruses, for their replication. Inthe absence of helper viruses AAVs integrate into the host cell genome,particularly at a specific site on chromosome 19.

The genome of AAVs is linear and has a length of about 4680 nucleotides.It comprises two reading frames which code for a structural gene and anon-structural gene. The structural gene is referred to as cap gene. Itis controlled by the P40 promoter and codes for three capsid proteins.The non-structural gene is referred to as rep gene and codes for the repproteins, Rep 78, Rep 68, Rep 52 and Rep 40. The two former proteins areexpressed under the control of the P5 promoter while the expression ofRep 52 and Rep 40 is controlled by the P19 promoter. The functions ofthe Rep proteins are determined inter alia by the control of replicationand transcription of the AAV genome.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an adeno-associated virus vector havinga foreign DNA which codes for a protein increasing the immunogenicity ofcells.

The present invention is based upon the finding that adeno-associatedviruses (AAVS) are suited to transduce tumor cells.

DETAILED DESCRIPTION OF THE INVENTION

An AAV vector according to the invention contains a foreign DNA whichcodes for a protein boosting the immunogenicity of cells.

The term “AAV vector” refers to any AAV, i.e., virus particle, and theDNA thereof. The AAV vector may be present in wild-type or modifiedform. The latter also means that it comprises expressible E4 sequences,particularly the ORF6 of the E4 sequences, of adenovirus. Moreover, itmay be a system of several components that provide individual or allfunctions of AAV and/or its DNA. Such a system comprises, e.g., arep-negative AAV vector and means providing an AAV Rep protein. The AAVvector may be inserted in cells where it integrates into the genome orremains in episomal form.

The term “foreign DNA coding for a protein boosting the immunogenicityof cells” encompasses any foreign DNA that may be integrated within anAAV vector and whose expression product can boost the immunogenicity ofcells. Examples of a foreign DNA are genes whose expression products arelacking from or are down-regulated in tumor cells, e.g., MHC-I genes,genes coding for costimulatory molecules, e.g., B7 genes, such as B7.1and B7.2 genes, genes coding for secretory immunostimulators, e.g.,cytokine genes such as IL-2, interferon and GM-CSF genes, and genes thatcode for tumor-associated antigens, e.g., MAGE1, tyrosinases or viralproteins, e.g., E7 protein of human papilloma virus and EBNA3 protein ofEpstein-Barr virus. It is preferred that the expression of the foreignDNA be controlled by a heterologous constitutive or inducible promotersuch as a tissue-specific or tumor-specific promoter. Furthermore, theforeign DNA can be inserted at any position in the AAV vector. In thisconnection, in some embodiments, the foreign DNA is present in or ispresent in place of the rep gene. In addition, in some embodiments,several foreign DNAs are present in an AAV vector.

Conventional methods may be used to prepare an AAV vector according tothe present invention. For example, an AAV vector can be prepared as avirus particle as follows: Two plasmids are provided wherein the firstplasmid is an AAV plasmid that contains a foreign DNA, e.g. a gene for aB7 molecule, between the 5′- and 3′-ITR sequences of AAV. However, thisplasmid referred to as pAAV-B7 does not code for the AAV Rep and AAV Capproteins. These proteins are encoded by a second plasmid. The secondplasmid contains an SV40 origin of replication. The second plasmid isreferred to as pSV40oriAAV. Both plasmids are transfected into cellsexpressing an SV40 T antigen. Such cells are, e.g., COS cells. The SV40origin of replication of pSV40oriAAV is activated by the T antigen, andthe plasmid is replicated. A high expression of the AAV Rep and AAV Capproteins is obtained. AAV vectors are obtained as virus particles byinfection of the COS cells with a helper virus, e.g., adenovirus. Thetiter is between 10⁶-10⁹ virus-particle/ml.

The immunogenicity of cells can be increased by means of such an AAVvector. In some preferred embodiments, an AAV vector coding for severalproteins boosting the immunogenicity of cells is provided. In some otherpreferred embodiments, several AAV vectors coding for differing proteinsboosting the immunogenicity of cells are provided. The increase inimmunogenicity can be achieved with cells of any kind, particularlytumor cells or pre-tumor cells such as HPV-transduced cervical cells.

The cells can be transduced with the AAV vector by conventional methods.If the AAV vector is present as a virus particle, the cells may beinfected with the viral particle. However, if the AVV vector is presentas DNA, it is advisable to transfect the cells therewith. For example,electroporation and lipofection may be used as transfection techniques.The cells can be present in an organism, or the cells to be transducedcan also be isolated from an organism, transduced outside the organismand be introduced into the organism again. Such cells are referred to asautologous cells. Moreover, allogenic cells may be used for thetransduction. In this connection, it is favorable for the allogeniccells to belong to a HLA type corresponding to the organism. The personskilled in the art readily understands methods of providing cells with acertain HLA type. In addition, it is preferred for the cells,particularly tumor cells or pre-tumor cells, to be inactivated beforeintroducing them again into the organism. Conventional methods such asirradiation may be used for this purpose.

Cells that are transduced outside an organism may also be co-cultivatedwith autologous and/or allogenic nuclear blood cells, particularlylymphocytes, based on the organism. The nuclear blood cells may bestimulated by this, and they may be introduced into the organism as suchor together with the transduced cells.

The present invention also relates to a vaccine that comprises an AAVvector according to the invention. The vaccine may further compriseconventional auxiliary agents such as buffers, diluents, carriers, etc.In some embodiments, the AAV vector codes for several proteins boostingthe immunogenicity of cells. In some other embodiments, several AAVvectors are provided that code for differing proteins boosting theimmunogenicity of cells. In some preferred embodiments, the vaccinecomprises further substances boosting the immunogenicity of cells,particularly tumor-specific antigens. These antigens may be present,e.g., in the form of peptides, particularly synthetic peptides. Theantigens may also be present in the form of expression plasmids encodingthem that can also code for HLA molecules. It is especially favorablefor the vaccine to also contain the cells transduced by the AAV vectorand/or the nuclear blood cells stimulated by these cells. In particular,it is preferred that the cells be inactivated. Moreover, it is preferredthat the vaccine contain a replication-defective adenovirus whose E4sequences are operational. Expressible E4 sequences may also be presenton a vector or an E4 protein. It is preferred that the vaccine contain asubstance supporting DNA replication. This may be, e.g., hydroxy urea, atopoisomerase inhibitor or a DNA synthesis inhibitor in a minor amount.It may also be favorable to irradiate the vaccine, e.g., X-ray or gammairradiation.

The present invention provides methods to transduce cells, particularlytumor cells or pre-tumor cells, more particularly freshly isolated tumorcells, with great efficiency. A transduction efficiency of 85 to 95% maybe obtained. Therefore, the most differing cells of a tumor may betransduced so that the entire antigen profile of the tumor is detectedand clonal selection is prevented. Moreover, transduction of theindividual cells may be achieved with a small number of AAV vectormolecules such as 10 to 20 molecules per cell. Therefore, thetransduction does not cause a cytopathic effect. Hence, the immunesystem recognizes the transduced cells and further cells having an equalantigen profile and can eliminate them.

Thus, the present invention is suited to boost the immunogenicity ofcells, particularly tumor cells or pre-tumor cells. As a result, thepresent invention is adaptable for use in vivo and/or ex vivo genetherapy of serious diseases such as cancers, including malignantmelanoma and cervical carcinoma.

The invention is further explained by the following examples. Theseexamples are provided only to further illustrate the present invention.

EXAMPLE 1 Preparation of an AAV Vector

2×10⁸ COS cells were added to 2.5 ml RPMI, in each case with 800 μg of a1:1 mixture of pSV40OriAAV and pAAV-B7 (cf. description above), andincubated in ice for 10 min. before electroporation was carried out in atotal volume of 0.5 ml. Thereafter, the cells were held on ice for 10min. before they were placed in tissue culture plates. The medium waschanged after 24 h and, after another 24 h, the cells were incubatedwith adenoviruses (10 infectious adenoviruses/COS cell) for 1 h. Afteranother 72 h, the cells were collected and pelleted. The cell pellet wasresuspended and homogenized. The homogenizate was adjusted with CsCl toa density of 1.4 g/cm³ and centrifuged at 38,000 rpm for 24 h. Fractionswere taken and those having an index of 1.375-1.371 were pooled,centrifuged and dialyzed against Tris buffer. The titer of the AAV virusparticles was determined.

EXAMPLE 2 Transduction of Tumor Cells with an AAV Vector

Primary melanoma tissue was isolated from human skin metastases. Themelanoma tissue was incubated in a 2% antibiotic solution(antibiotic/antimycotic agent) for 2×30 min. before it was cut intosmall fragments. The fragments were passed through a metal screen, andthe resulting cell suspension was pipetted through a fine screen into acentrifuge tube. Erythrocytes and dead cells were removed via a Ficollgradient before the living melanoma cells were washed 2 × with PBS. Themelanoma cells were then incorporated into a suitable medium (RPMI 1640,1% glutamine, 10-20% fetal calf serum, 1% penicillin streptavidin) andplaced in tissue culture plates. Prior to the transduction, the melanomacells were characterized by means of antibodies (S100, HNB-45, companyof DA-KO).

3×10⁵ melanoma cells were placed in a 24-well tissue culture plate.3×10⁶ AAV virus particles of Example 1, which code for a B7 molecule,were placed in 250 μl serum-free medium on the melanoma cells. After anincubation period of 1 h, 1.5 ml serum-containing medium were added, andthe cells were incubated in an incubator.

A FACS analysis of the B7 transduction was carried out. Mouse-anti-humanB7.2 monoclonal antibodies (Pharmingen) were placed on the cells whichwere then bound with a FITC-conjugated goat-anti-mouse antibody.Non-specific bonds were ruled out by isotypic controls. A minimum of10,000 living melanoma cells was used for each analysis. The percentageof positive cells was defined as fraction which are beyond the 99% rangeof the control group.

A B7 transduction efficiency of 85-90% was obtained. Parallelexperiments in which an AAV virus particle that coded for severalproteins boosting the immunogenicity of cells and in which several AAVvirus particles that coded for differing proteins boosting theimmunogenicity of cells, respectively, was used for the transduction ofthe tumor cells, showed equal transduction efficiencies for theindividual proteins.

EXAMPLE 3 Stimulation of the Immunogenicity of Cells by an AAV Vector

(a) The stimulation of the immunogenicity of cells was determined bymeans of inducing a cytotoxic T cell response.

Melanoma material was removed from various tumor patients. At the sametime, blood was taken, and the T cells were accumulated. As describedabove, the tumor cells were isolated and transduced with the AAV virusparticles of Example 1. Thereafter, the T cells were stimulated with thetransduced tumor cells. Non-transduced tumor cells were used for thestimulation of the T cells as control.

The results demonstrated that the activity of the cytotoxic T cells canmarkedly be increased by stimulation with an AAV virus particleaccording to the invention.

In another experiment, a primary tumor was removed from a tumor patientand treated as described in Example 2. 1×10⁶ tumor cells were seeded andinfected with a multiplicity of infection of 10 after previous gammairradiation by 30 Gy with B7.1-AAV and GM-CSF-AAV. Four days after theinfection, the expression rate of the foreign genes was determined forB7.1 and GM-CSF by means of Facs-Flow and ELISA, respectively. 20 ml ofperipheral blood was withdrawn from the patient. Peripheral bloodlymphocytes were isolated by density gradient centrifugation usingFicoll Histopaque. 1×10⁷ lymphocytes were co-cultivated in RPMI1640medium, containing 10% heat-inactivated human serum, 2 mM glutamine,non-essential amino acids, 2 mM sodium pyruvate and 100 μg/ml gentamycinor kanamycin, with the AAV-infected tumor cells to stimulate atumor-specific immune response by cytotoxic T cells. After one week ofco-cultivation, 20 U/ml recombinant human interleukin-2 was added to themedium. At intervals of 10 days, further tumor cells infected with theAAV vectors were added to the culture in a ratio of 1:10 to thelymphocytes. After three weeks of cultivation, the lymphocytes werechecked in a cytotoxicity test (chromium release test or europiumrelease test) for their capability of lyzing the tumor cells. 1×10⁷lymphocytes were reinfused intravenously into the patient.

(b) C57/B16 mice were infected with 5,000 living B16-F10 tumor cellseach (mouse melanoma model) into the caudal vein. These tumor cells ledto the formation of metastases that could be detected after about 20days by obduction predominantly in the liver and lungs. On days 3, 10and 17 following the tumor cell injection, part of the mice wereimmunized with 300,000 B16-F10 tumor cells that had previously beentransduced with an AAV virus particle coding for a B7 protein. Thus, themolecule B7 was found on the surface of the tumor cells.

It showed that the formation of metastases can be inhibited byimmunization with an AAV virus particle according to the invention.Parallel experiments in which an AAV virus particle which coded forseveral proteins boosting the immunogenicity of cells and in whichseveral AAV virus particles that coded for differing proteins boostingthe immunogenicity of cells, respectively, was used for the transductionof the tumor cells, showed an even more intense inhibition of theformation of metastases.

(c) C57/B16 mice were injected subcutaneously in each case 100,000B16-F10 tumor cells into the back. This resulted in the formation of atumor that had a circumference of 0.3-0.5 cm after 10 days. At thattime, the AAV virus particle of Example 1 (10⁶ -10⁸ particles) wasinjected directly into the tumor.

The results demonstrate that after the transduction with an AAV virusparticle according to the invention, the tumor cells are detected by theimmune system and the tumor is eliminated.

What is claimed is:
 1. A method for producing tumor cells with increasedimmunogenicity for treating tumor patients comprising the steps ofobtaining freshly isolated tumor cells and/or pre-tumor cells, andtransducing said cells with an Adeno-Associated Virus vector comprisinga foreign DNA coding for a protein that boosts the immunogenicity of acell.
 2. The method according to claim 1, wherein the foreign DNAcomprises a gene selected from the group consisting of a gene whoseexpression product is lacking or is down-regulated in said tumor cells,a gene coding for a co-stimulatory molecule, a gene coding for asecretory immunostimulator, and a gene coding for a tumor-associatedantigen and viral protein.
 3. The method according to claim 1, whereinthe AAV vector comprises more than one foreign DNAs.
 4. The methodaccording to claim 1, wherein the foreign DNA is controlled by aheterologous constitutive or inducible promoter.
 5. The method accordingto claim 4, wherein the promoter is a tissue-specific or tumor-specificpromoter.
 6. The method according to claim 1, wherein said AAV vector isin the form of a vaccine which contains conventional auxiliary agents.7. The method according to claim 6, wherein the vaccine comprises morethan one AAV vector and each AAV vector codes for a different proteinthat boosts the immunogenicity of a cell.
 8. The method according toclaim 6, wherein the vaccine further comprises substances that boost theimmunogenicity of a cell.
 9. The method according to claim 8, whereinthe substances are tumor-specific antigens.
 10. The method according toclaim 6, wherein the AAV vector is present in freshly isolated tumorcells.